Ligands for capturing microvesicles and uses thereof

Abstract

The invention relates to ligands able to bind to microvesicles, comprising a plurality of metal(II)-dipicolylamine moieties and their use in the diagnosis or prognostic of disorders in a subject.

1 - 23 . (canceled) 
     
     
         24 . An in vitro method for capturing microvesicles present in a sample, which comprises contacting the sample with a ligand able to bind to microvesicles, said ligand comprising at least one moiety of formula (I): 
       
         
           
           
               
               
           
         
         wherein M is a metal cation selected from the group consisting of Zn 2+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+  and Fe 2+ , in conditions suitable to form a complex between said ligand and said microvesicle. 
       
     
     
         25 . The in vitro method of  claim 24 , wherein the ligand comprises at least one moiety of formula (Ia) as follows: 
       
         
           
           
               
               
           
         
         wherein:
 each X 1  is independently O, S, or NH, 
 each X 2 , when present, is independently O, S or NH, 
 M is selected from the group consisting of Zn 2+ , Co 2+ , Cu 2+  and Fe 2+ . 
 
       
     
     
         26 . The in vitro method of  claim 25 , wherein the ligand comprises the moiety of formula (Ib) 
       
         
           
           
               
               
           
         
         wherein:
 each X 1  is independently O, S, or NH, 
 each X 2 , when present, is independently O, S or NH, 
 M is selected from the group consisting of Zn 2+ , Co 2+ , Cu 2+  and Fe 2+ . 
 
       
     
     
         27 . The in vitro method of  claim 24 , wherein the ligand is a dendrimer comprising a branched core bearing a plurality of moieties of formula (I), said branched core comprising a group selected from the group consisting of 3,5-di(hydroxymethyl) phenol, 3,5-di(thiomethyl)phenol, 3,5-di(thiomethyl) thiophenol, 3,5-dialkylphenol, 3,5-di(aminomethyl)phenol, 3,5-dialkylphenol and 3,5-di(aminomethyl) phenylamine as building block. 
     
     
         28 . The in vitro method of  claim 24 , wherein the ligand further comprises a mean for immobilization on a support attached at the extremity of a spacer chain. 
     
     
         29 . The in vitro method of  claim 28 , wherein the ligand is of formula (II) 
       
         
           
           
               
               
           
         
         wherein:
 n is an integer from 1 to 10, 
 m, p and o are independently 0 or 1, 
 M is a divalent metal cation, 
 [CORE] is a chemical entity bearing the at least one moiety of formula (I) and having a molecular weight of at most 20 000 g·mol −1 , 
 [IMM] is a mean for covalently or non-covalently immobilization on a support, and 
 [SPACER] is selected from the group consisting of a peptide, a polypeptide, an oligo- or polysaccharide, a saturated or unsaturated hydrocarbon chain optionally interrupted by one or several heteroatoms, optionally having an heteroatom on at least one of its ends, and optionally substituted by one or several substituents selected from hydroxyl, halogens, C 1 -C 3  alcoxy, —CN, —CF 3 , or C 1 -C 3  alkyl, polymers including homopolymers, copolymers and block polymers, or combinations thereof. 
 
       
     
     
         30 . The in vitro method of  claim 29 , wherein the ligand is further characterized by one, several or all of the following features:
 m, p and o are 1,   M is selected from the group consisting of Zn 2+ , Co 2+ , Cu 2+  and Fe 2+ ,   [SPACER] is selected from:
 C 2 -C 20  saturated or unsaturated hydrocarbon chains optionally substituted, and optionally having an heteroatom on at least one of its ends; polyamide chains comprising from 2 to 20 monomers; polyester chains comprising from 2 to 10 monomers and polyether chains comprising from 2 to 20 monomers and combinations thereof, 
   [IMM] comprises, or consists of, a moiety selected from the group consisting of an amino group, OH, —COOH, an activated carboxylic acid, —SH, iodoacetyl group, a carbonyl, a hydrazide group, an azido, and a strained alkyne and   [CORE] is a chemical moiety made of at least one 3,5-di(hydroxymethyl) phenol.   
     
     
         31 . The in vitro method of  claim 29 , wherein the ligand is selected from compounds of formula (IIb) and (IIc): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein
 m, p and o are independently 0 or 1, 
 M is a divalent metal cation, 
 [IMM] is a mean for covalently or non-covalently immobilization on a support, 
 [SPACER] is selected from the group consisting of a peptide, a polypeptide, an oligo- or polysaccharide, a saturated or unsaturated hydrocarbon chain optionally interrupted by one or several heteroatoms, optionally having an heteroatom on at least one of its ends, and optionally substituted by one or several substituents selected from hydroxyl, halogens, C 1 -C 3  alcoxy, —CN, —CF 3 , or C 1 -C 3  alkyl, polymers including homopolymers, copolymers and block polymers, or combinations thereof, 
 each X 1  and X 2 , when present, are selected from the group consisting of O, NH, and S, 
 X 3  is selected from the group consisting of —O—, C(═O) —OC(O)—, —C(O)O—, —OC(O)O—, —S—, —SS—, —SC(O)—, —OC(S)—, NR 1 —, —NR 1 C(O)—, —C(O)NR 1 —, NR 1 C(S)—, C(S)NR 1 —, —OC(O)S—, —OC(S)O—, —SC(O)O—, —OC(S)S—, —SC(O)S—, —SC(S)O—, —SC(S)S—, OC(O)NR 1 —, —OC(S)NR 1 —, —NR 1 C(S)O—, —NR 1 C(O)S—, NR 1 C(O)NR 2 —, —NR 1 C(S)NR 2 —, —SC(O)S—, —SC(S)O—, —S(O)—, —S(O) 2 —, —P(O)(R 1 )—, —P(O)(OR 1 )—, P(O)(R 1 )O—, OP(O)(OR 1 )—, OP(O)(R 1 )O—, NR 1 P(O)(R 2 )—, —NR 1 P(O)(OR 2 )—, NR 1 P(O)(R 2 )O—, OP(O)(OR 1 )— and OP(O)(R 1 )O— wherein R 1  and R 2  are independently H or CH 3 . 
 
       
     
     
         32 . The in vitro method of  claim 31 , wherein the ligand of formula (IIb) or (IIc) is such that:
 m and p are 1,   M is Zn 2+ , Co 2+ , Cu 2+  or Fe 2+ ,   X 1  and X 2 , when present, are O,   X 3  is O, NH, NHCO, CONH, O(C═O), (O═C)O, O(C═O)O, or NHCONH,   [IMM]-[SPACER] is NH 2 —(CH 2 ) r — or NH 2 —[(CH 2 ) 2 —O] r — with r an integer from 2 to 10, and   said molecule comprises one or several counter-anions selected from the group consisting of perchlorate, tosylate, nitrate, sulphate, sulphonate, thiosulfate, halide, hexafluorophosphate, tetraphenylborate, carbonate, and tetrafluoroborate.   
     
     
         33 . The in vitro method of  claim 31 , wherein the ligand is selected from compounds of formula (IIIb) and (IIIc): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         34 . The in vitro method of  claim 24 , wherein the ligand is immobilized on a support. 
     
     
         35 . The in vitro method of  claim 24 , which further comprises at least one step selecting from:
 a step of providing a sample susceptible to contain the microvesicles of interest from a body fluid of a subject, and/or   a step of washing the support on which the ligand is immobilized after the step of contacting the ligand with the sample, and/or   a step of detecting the formation of the complex between the microvesicles of interest and said ligand, and/or   a step of recovering the complex formed between the microvesicles and the ligand, and/or   a step of quantification of said microvesicles of interest, and/or   a step of characterization of the captured microvesicles by detecting and/or quantifying a biomarker present on the surface or within the microvesicles, and/or   a step of releasing the microvesicles from the complex, and/or   a step of recovering the microvesicles of interest.   
     
     
         36 . An in vitro method for the diagnosis, the differential diagnosis, the prognosis, the assessment of the risk of, and/or the monitoring a disorder in a subject which comprises:
 (a) providing a sample from the subject susceptible to contain microvesicles,   (b) capturing the microvesicles possibly present in the sample by carrying out the method of  claim 24 , and   (c) detecting, quantifying and/or characterizing the microvesicles captured in step (b) by complexation.   
     
     
         37 . The in vitro method of  claim 36 , wherein:
 the disorder is selected from the group consisting of diabetes, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, and diabetic foot syndrome, multiple sclerosis, cancer, Alzheimer's disease, Parkinson's disease, aneurysm, cerebral vasospasm, stroke, and coronary artery disease, and/or   the sample is, or derives from, blood, blood plasma and urine.   
     
     
         38 . A molecule able to bind to microvesicles and comprising at least one moiety of formula (Ia) as follows: 
       
         
           
           
               
               
           
         
         wherein:
 each X 1  is independently O, S, or NH, 
 each X 2 , when present, is independently O, S or NH, 
 M is selected from the group consisting of Zn 2+ , Co 2+ , Cu 2+  and Fe 2+ . 
 
       
     
     
         39 . The molecule of  claim 38 , which comprises a moiety of formula (Ib) 
       
         
           
           
               
               
           
         
         wherein:
 each X 1  is independently O, S, or NH, 
 each X 2 , when present, is independently O, S or NH, 
 M is selected from the group consisting of Zn 2+ , Co 2+ , Cu 2+  and Fe 2+ . 
 
       
     
     
         40 . The molecule of  claim 38 , which is a dendrimer comprising a branched core bearing a plurality of moieties of formula (I), said branched core comprising a group selected from 3,5-di(hydroxymethyl) phenol, 3,5-di(thiomethyl)phenol, 3,5-di(thiomethyl) thiophenol, 3,5-dialkylphenol, 3,5-di(aminomethyl)phenol, 3,5-dialkylphenol and 3,5-di(aminomethyl) phenylamine as building block. 
     
     
         41 . The molecule of  claim 38 , which further comprises a means for immobilization on a support attached at the extremity of a spacer chain. 
     
     
         42 . The molecule of  claim 41 , which is of formula (IIb) and (IIc): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein
 M, m, p, [IMM] and [SPACER] are as defined above for formula (II), 
 each X 1  and X 2 , when present, are selected from the group consisting of O, NH, and S, 
 X 3  is selected from the group consisting of —O—, C(═O) —OC(O)—, —C(O)O—, —OC(O)O—, —S—, —SS—, —SC(O)—, —OC(S)—, NR 1 —, —NR 1 C(O)—, —C(O)NR 1 —, NR 1 C(S)—, C(S)NR 1 —, —OC(O)S—, —OC(S)O—, —SC(O)O—, —OC(S)S—, —SC(O)S—, —SC(S)O—, —SC(S)S—, OC(O)NR 1 —, —OC(S)NR 1 —, —NR 1 C(S)O—, —NR 1 C(O)S—, NR 1 C(O)NR 2 —, —NR 1 C(S)NR 2 —, —SC(O)S—, —SC(S)O—, —S(O)—, —S(O) 2 —, —P(O)(R 1 )—, —P(O)(OR 1 )—, P(O)(R 1 )O—, OP(O)(OR 1 )—, OP(O)(R 1 )O—, NR 1 P(O)(R 2 )—, —NR 1 P(O)(OR 2 )—, NR 1 P(O)(R 2 )O—, OP(O)(OR 1 )— and OP(O)(R 1 )O— wherein R 1  and R 2  are independently H or CH 3 . 
 
       
     
     
         43 . The molecule of  claim 42 , wherein the ligand of formula (IIb) or (IIc) is such that:
 m and p are 1,   M is Zn 2+ , Co 2+ , Cu 2+  or Fe 2+ ,   X 1  and X 2 , when present, are O,   X 3  is O, NH, NHCO, CONH, O(C═O), (O═C)O, O(C═O)O, or NHCONH,   [IMM]-[SPACER] is NH 2 —(CH 2 ) r — or NH 2 —[(CH 2 ) 2 —O] r — with r an integer from 2 to 10, and   said molecule comprises one or several counter-anions selected from the group consisting of perchlorate, tosylate, nitrate, sulphate, sulphonate, thiosulfate, halide, hexafluorophosphate, tetraphenylborate, carbonate, and tetrafluoroborate.   
     
     
         44 . The molecule of  claim 43  which is selected from compounds of formula (IIIb) and (IIIc): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         45 . A support having thereon a ligand as defined in  claim 38 .